Cancer is the second most common disease and also one of the most feared. Cancer occurs when cells continue to divide and fail to die at the appropriate time. Under normal circumstances, the many types of cells that make up the body grow and divide to produce more cells as they are needed in order to maintain a healthy body. Tumors may form when this orderly process is disrupted by changes in genes that control normal cell growth and death and cellular growth becomes uncontrolled. Genetic changes that arise internally due to defective DNA repair or may be induced by external factors such as diet, exposure to ultraviolet or other types of ionizing radiation, viruses such as cervical papillomaviruses, exposure to chemical carcinogens in the workplace or in the environment, drug or tobacco use, or to agents such as asbestos. Some detrimental genetic alterations are inherited.
Regardless of which particular combination of insults and accidents contribute to the root cause of cancer, cumulative mutations in cells can arise from alterations in specific locations in the DNA of the cell. Pieces of chromosomes may become scrambled, truncated or fused together; or entire chromosomes may be lost or duplicated in their entirety. As a result of these alterations cancer cells proliferate more rapidly than neighboring normal cells. The cell abnormalities are passed down to their cellular descendants as these clonal armies continue to grow unabated. They eventually develop the capacity through additional mutations to invade and destroy surrounding tissue. Malignant cancers usually become life-threatening because they develop the power to disable the regulatory mechanisms that confine them to the specific tissue in which they arose so that they disengage from the malignant tumor and travel through the bloodstream or lymphatic system where they eventually interfere with vital systems.
Current Cancer Therapies
New and effective cancer treatments are constantly being sought. The most common therapies include radiation and drug treatments, many of which are toxic and harmful to normal cells. Conventional therapies may kill the majority of cells within a tumor but a small number of unaffected cells may be able to reestablish the aberrant pattern of proliferation.
While most malignant cells appear to be highly susceptible to current cancer treatments, there is some speculation that a certain subset of cells, perhaps stem cells, are more resistant to drugs and radiation than normal, non-cancerous cells. Alternatively, tumor cells may simply develop resistance to chemical and radiation treatments, leading to recurrence of chemo- and/or radio-resistant cancers because the resistant cells maintain their ability to proliferate indefinitely. Resistance may also develop because administration of chemotherapeutic agents for the treatment of tumors is restricted by the toxicity of these agents to normal cells.
Gene Therapy
One approach to lowering toxicity of high drug doses is transfection of healthy, normal stem cells with transgenes that confer resistance to these agents. In theory, this will result in cytotoxic drug-resistant cells and allow the administration of higher, therapeutically significant doses of chemotherapeutic agents. Use of transfected cells has been suggested for protection of bone marrow cells since bone marrow cells are rapidly dividing and thereby most at risk to chemotoxicity and in fact has shown some success in animal models, Licht et al., 2000.
Recently, gene therapy methods have been explored both as cancer diagnostics and as cancer treatments. Because most forms of cancer are complex, multifactorial, and multigenic in nature, many conceptual and technical obstacles remain to be overcome in order to approach this disease at the genetic level. Yet, it is the molecular nature of tumorigenesis, i.e., the activation of dominant oncogenes and/or the inactivation of tumor suppressor genes, which provides insight for such strategies in that these genetic events represent novel targets for molecular therapy. Genetic analysis is already being used in diagnostic and prognostic predictions in certain malignancies; e.g., amplification of erb-B2 in breast and ovarian cancer; amplification of N-myc in neuroblastoma; and ras mutations in adenocarcinoma of the lung.
Unfortunately, the list of oncogenes and tumor suppressor genes continues to grow. Recently, mutations in at least 15 tumor suppressor genes were identified and the number of oncogenes now exceeds 100 (Gibbs, et al., 2004). The consequences remain uncertain, but the evidence points to the possibility that each tumor may be unique in its genetic disarray. If this is true, the prospect of tracking the root causes, categorizing the distinguishing marks of a particular cancer as well as determining early diagnoses and accurate prognostic evaluations become exponentially more difficult, and the chance even more remote of finding a treatment specific for each tumor.
Compounding this complexity, researchers are now finding that whereas an individual tumor may begin its life of malignancy with a particular accumulation of mutations in a single cell, which then passes down its genetic abnormalities to its descendants, the tumor itself is comprised of a large diversity of cells. Although all these cells are very different from normal human cells, they are not the masses of identical clones as once believed. In fact, there seems to be a small subset of cells within each tumor that is responsible for not only the growth of the tumor but for its metastasis.
Despite some progress in developing gene therapy methods, use of these methods in the treatment of cancer still has several obstacles to overcome. In vivo treatments for malignant melanoma in dogs, for example, has met with some success, showing a positive response to tumor regression over a period of 6-12 weeks after a direct DNA injection encoding a Staphylococcus antigen and GM-CSF cytokine (WO96/36366). Liposome/Staphylcoccal antigen injections alone, however, failed to show any effect even after 17 weeks, suggesting that tumor regression was caused by a toxic effect generated by the cytokine or cytokine/antigen combination in the cancer cells.
The use of “informational drugs,” a type of gene therapy, has also been proposed. Antisense oligonucleotides, small synthetic nuclease-resistant nucleotide sequences complementary to specific RNA sequences, are an example of this type of drug. By specifically binding and thereby inhibiting transcription and/or translation of a single oncogene, it may be possible to block oncogenesis and even reverse clinical symptoms. Unfortunately, the efficacy of informational drugs seems to depend on their use with other drugs. Limited effectiveness has been observed in a study of melanoma patients using an antisense molecule in combination with dacarbazine. Some progress is being made in targeted antisense oligonucleotide therapy; for example, it was reported that therapeutic effectiveness of the cancer therapeutic agent trinotecan in mice was increased by administration of an antisense molecule tarteted to RIalpha subunit of camp-dependent protein kinase (Wang, et al., Int. J. Oncol. Jul. 21, 2002 (1), 73-80). However, antisense application has yet to live up to the expectation of being widely applicable for all types of cancers.
Immunotherapy
The manipulation of the host immune system to identify cancer cells as non-self; i.e., methods to mobilize and strengthen the immune system so that it can selectively destroy and/or inhibit proliferation of cancerous cells, is gaining more attention. This is due to the recognition that the host itself may be able to generate the safest and most effective defense against cancer.
Large numbers of people are exposed to carcinogens every day; yet only a tiny minority suffer life-threatening tumors. Given so many opportunities for aberrant cells to arise, it is remarkable that most hosts do not inevitably succumb to cancer. Clearly, the most effective solution is to eliminate cancerous cells before they have the chance to cycle out of control.
Immunologists are in general agreement that the body is capable of protecting itself from cancer. Historically, it was postulated that the transformation of normal tissues into neoplasms was accompanied by the expression of new molecules, i.e., tumor-specific antigens. Early studies attempted to prove the existence of tumor-associated antigens by transplanting tumors from one animal to another and found that the tumors were rejected. The rejection was wrongly attributed to the presence of neo-antigens instead of disparate histocompatibility antigens, as is known today.
The vast majority of malignancies arise in immunocompetent hosts, raising doubts as to whether a general strengthening of the immune system can ever be effective in targeting cancer cells, which are not always recognized as foreign by the host. It is now known that tumor cells do indeed carry antigens that are different from their normal counterparts. These antigens can be tumor-associated or tumor-specific. Tumor-associated antigens can be generated by the activation of normally repressed genes, such as oncofetal antigens which are normally synthesized during embryogenesis but are not found on adult cells; some are present but are masked; some molecules may be lost when the cells become transformed and thus alter the profile of adjacent molecules by their absence; some antigens may be modifications of normal molecules; and some may be nuclear or cytoplasmic and thus hidden from immune surveillance. Tumor-specific antigens are restricted to tumor tissues. They are not found in normal adult or fetal tissues and are rare.
In order for an immunotherapy to be effective, evasive techniques used by tumor cells must be overcome. Some therapies under investigation administer chemical messengers such as cytokines like IL-2 and IL-12 alone or in combination, lymphocytes specific for telomerase, bacterial extracts from Corynebacterium granulosum as adjuvants, or drugs which boost the immune system. These are aimed at heightening the immune response in general. In an attempt to make the immune response more specific for the tumor cells, some employ the use of autologous tumor cells, either combined with cytokines such as GM-CSF, gamma interferon or IL-2, individually or in combination, or transfected with the genes that encode these cytokines. A similar approach utilizes tumor cell lines instead of autologous tumor cells.
Antigens, bacterial and viral, have also been used in combination with cytokine or other immunomodulator genes delivered by means of adenovirus, retrovirus or plasmid vectors (WO 94/21808; WO 96/29093). The presence of cytokines is a factor in the relative success of some of these approaches. In some cases, a highly destructive and specific response to otherwise nonimmunogenic tumors can be elicited by the insertion of genes encoding interleukin-2, interleukin-4, interleukin-12, interferon-γ, interferon-α and/or tumor necrosis factor into the tumor cells as well as into cytotoxic lymphocytes or macrophages, although can serious side-effects can be caused at high doses.
Several approaches use bio-signals to direct immune effector cells to the tumor in a non-specific manner. Other approaches focus on the immune cells themselves. Autologous antigen presenting cells, including dendritic cells have been loaded with tumor, with cytokines and/or with total tumor RNA in an effort to make the immune response more specific for the tumor (Cranmer, et al., 2004).
Oncophages have been used to lyse autologous tumor cells in the hope of generating a tumor-specific response or have transfected tumor cells with immunotoxins (Wallack, et al., 1995). Patients also have been vaccinated with specific tumor antigens, tumor-specific monoclonal antibodies, HSP 70 purified from autologous tumor cells, autologous T cells activated against tumor cells ex vivo. These methods focus on specific aspects of the immune response to particular tumor characteristics.
Some immunotherapeutic modalities are based on studies in which tumor cells are transfected with genes encoding Major Histocompatibility Complex (MHC) (Hock et al., 1996; EP 569678; WO 95/13092), Calmette-Guérin (BCG) (Morton, et al., 1992) and Mycobacterium (Menard et al., 1995) antigens. However, despite promising results when compared with control groups, the significant survival advantage conferred by systemically administered antigen such as BCG was not confirmed in concurrently controlled randomized clinical trials.
Autologous tumor-infiltrating lymphocytes have been used in genetic immuno-modulation studies because of their inherent specificity for the tumor and their ability to home back to the tumor site when reinfused into the patient. Normal tissue has been protected by stably transfecting normal bone marrow cells with cytokine genes prior to chemotherapy, thereby achieving a more continuous effect while obviating the need to infuse drugs which have short half-lives and produce systemic side effects when delivered intravenously (Yamaguchi, et al., 2003).
T-lymphocytes recognize at least two different types of antigens; peptides derived from conventional protein antigens, and the so-called “superantigens”. The classical definition for superantigen is a polypeptide that reacts in some ways like conventional antigens but exhibits critical differences in others (Johnson et al., 1992). Before a T helper cell can recognize conventional protein antigens, these proteins must first undergo processing by macrophages or other antigen presenting cells (APCs). APCs then display the peptide on the cell surface in combination with MHC. Unlike typical antigens, however, the distinguishing feature of superantigens according to Johnson et al., (1992) is the ability to bind MHC directly, to specific Vβ segments of TCR that are outside of the normal antigen-binding groove, without uptake and processing by APCs. Some examples of superantigens include the soluble exotoxins produced by gram-positive bacteria such as Staphylococcus aureus, which typify bacterially-derived superantigens, and viral superantigens such as those encoded by endogenous mouse mammary tumor viruses.
Conjugation between the superantigen staphylococcal enterotoxin-A (SEA), and mAbs recognizing human colon cancer enables T cells to lyse colon carcinoma cells in vitro (Giantonio, et al., 1997). Staphylococcal enterotoxins have been suggested as possible cancer vaccine candidates (WO 95/0178). However, there is no evidence that transfected cells produce a sufficient in vivo immune response in human cancer patients. Likewise, induction of a T-cell response is described in WO 96/36366 where after repeated administration, genes encoding SEA and a cytokine were effective in causing regression or slowing growth of a canine melanoma of the composition. Administering the superantigen alone was ineffective.
Deficiencies in the Art
No general method for treating cancers has yet been developed or have productive methods been suggested. The search continues to find treatments that cure and/or arrest and prevent the many types of tumors and leukemias that affect individual health as well as economic issues. Advantages of immunotherapy are considerable when compared to standard treatments in that little or no toxicity has been seen in clinical trials thus far, and vaccine-induced regression, when achieved, is usually durable, often lasting from months to years. However, past efforts to marshal host defenses by stimulating an immune response to specific cancers have generally failed, despite use of immunostimulatory and gene therapy methods.
Therapies are needed that will effectively address controlling and/or curing of a wide range of cancers and do not suffer some of the disadvantages of high toxicity encountered with current radiation and chemotherapy regimes. An effective cancer vaccine, for example, must elicit both humoral (antibody) and appropriate cellular (antigen-specific T cell) responses. While some viral antigens expressed in tumor cells activate both these arms of the immune system, the response is so rapid that the genetically modified tumor cells were eliminated before T cells reactive to the tumor itself could be developed. Despite decades of effort and dozens of different approaches, no immunotherapy has emerged as a standard therapy for any type of cancer. There is therefore an unmet need to find ways to control and eliminate cancer cells in vivo without toxic effects and provide permanent protection against a wide range of cancers.